This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. Heme is essential in almost every single aspect of cell function, as it is a required prosthetic group in proteins with very diversified functions. Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the insertion of ferrous ion into protoporphyrin IX forming the product protoheme IX and has been recognized as a key enzyme in heme biosynthesis since 1956. Unfortunately, for the past decades, research on ferrochelatase has been hindered by the difficultly in obtaining sufficient quantities of purified active enzyme, in part due to the association of ferrochelatase with membranes and the low abundance of ferrochelatase in mitochondria. Most of our knowledge concerning the active sites of ferrochelatase was derived from kinetic studies of chemically modified ferrochelatase and inhibition studies. More direct physical investigations, such as spectroscopic studies, were not possible due to the limited availability of purified proteins. This major hurdle has been overcome by one of our collaborators, Dr. G. C. Ferreira, who has successfully overexpressed the murine ferrochelatase in Escherichia coli. Most importantly, the recombinant enzyme is associated with the bacterial soluble fraction, facilitating the subsequent purification and manipulation of the enzyme. We now have essentially unlimited quantities of purified active ferrochelatase for spectroscopic and kinetic studies. Our preliminary EPR and Mossbauer spectroscopic studies of the recombinant enzyme reveal an important and surprising result. That is, mammalian ferrochelatase is a metalloenzyme containing a [2Fe-2S] cluster. These new developments have opened up many new avenues for ferrochelatase research. We therefore propose to use a combination of spectroscopic, chemical, biochemical, and molecular biological approaches to investigate the structure and function of ferrochelatase. In particular, we propose (1) to apply Mossbauer, EPR and NMR spectroscopy to characterize and to obtain structural information about the ferrous binding site and the [2Fe-2S] cluster, (2) to identify the amino-acid residues essential for ferrochelatase function using site-directed mutagenesis in combination with spectroscopic and kinetic investigations, and (3) to elucidate the mechanism of ferrochelatase employing rapid freeze-quench EPR and Mossbauer techniques. Since ferrochelatase uses iron as a substrate and our preliminary results established that it is an iron-containing enzyme, the proposed spectroscopic approach, a combination of Mossbauer and EPR techniques, is particularly suited for the studies of ferrochelatase. The proposed investigations are expected to yield characteristic physical properties and structural information concerning the substrate iron binding site and the 2[Fe-2S] clusters, and to provide detailed mechanistic information. Possible reaction intermediates may be trapped and characterized, and its rates of formation and decay will be determined. the proposed studies are also expected to shed light on the functional role of the [2Fe-2S] cluster in ferrochelatase and provide new insights in general into the role of iron sulfur clusters in proteins.